Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

Abstract

The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a beta-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360 degrees clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP-PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors.

Figure 1.

Sequence alignment of RecA family proteins from S.solfataricus (Sso RadA), M.voltae (MvRadA), P.furiosus (PfRad51), H.sapiens (HsRad51 and HsDmc1), S.cerevisiae (ScDmc1 and ScRad51) and E.coli (EcRecA). All these RecA-like strand exchange proteins have similar N-terminal domains. The C-terminal RecA domains have been removed for clarity. Secondary structural features of the left-handed SsoRadA helical filament are indicated in cyan (α helices) and red (β strands). Functional motifs are indicated under their corresponding amino acid sequences: the putative dsDNA binding HhH motif, the putative ssDNA binding L1 and L2 loops, the ATP binding Walker A and B motifs, the polymerization motif (PM), the subunit rotation motif (SRM), and others. Positions of the R₀–E₁–E₂ triad are indicated using blue arrows.

Figure 2.

EM images of the wild type SsoRadA left-handed helical filaments. SsoRadA protein (1 mM) was incubated at 65°C for 5 min in a D-loop formation reaction buffer [1 mM AMP–PNP, 2 mM MgCl₂, 0.5 mM DTT, 10 mM HEPES pH 7.0, 5 μM linear ssDNA (500 nucleotides in length), 5 μM dsDNA (2000 base paris in length), 50 mg/ml bovine serum albumin], and chilled on ice to stop the reaction. The reaction product was diluted 100-folds with EM sample dilution buffer (1 mM AMP–PNP, 2 mM MgCl₂, 0.5 mM DTT, 10 mM HEPES pH 7.0). The right- and left-handed helices are indicated by arrow, respectively.

Figure 3.

MvRadA-AMP-PNP, PfRad51 and SsoRadA quaternary structures. Side and top views are shown as indicated. The helical pitches of the right-handed MvRadA-AMPPNP filament, the left-handed SsoRadA filament and the overwound right-handed SsoRadA filament are 106.7 Å, 125.6 Å and 98.0 Å, respectively. The putative dsDNA binding regions or HhH motifs are highlighted in blue. The putative ssDNA binding L1 and L2 motifs are highlighted in pink and green, respectively. The helical filament axis is depicted as an orange rod running through the filament.

Figure 4.

MvRadA-AMP-PNP, PfRad51 and SsoRadA monomer structures. The N-terminal domain (NTD, in cyan), the conserved phenylalanine residues (in yellow), polymerization motif (PM, in pink) and α5 helix (in blue) are indicated. The subunit rotation motif (SRM) is located at the C-terminal of the α5 helix. Two α helices (α10 and α12) at the core ATPase domain (CAD) are shown in green to illustrate subunit rotation.

Figure 5.

Subunit rotation influences the quaternary structures of archaeal RadA and Rad51 proteins. The structures of the left-handed SsoRadA helical filament (in cyan), the PfRad51 closed ring (in red), the right-handed MvRadA-AMPPNP helical filament (in green) and the overwound right-handed SsoRadA helical filament (in pink) are superimposed by fixing the N-terminal domain (NTD, in yellow) and polymerization motif (PM). The side chain of phenylalanine residue of PM is indicated in green. The Core ATPase domain (CAD) of neighboring protomers is indicated in gray. The subunit rotation motif (SRM) locates immediately after the α5 helix. AMP-PNP in the right-handed MvRadA-AMPPNP helical filament is also marked by arrow, respectively. The disposition of adjacent α11 and α12 helices are shown to illustrate subunit rotation.

Figure 6.

Subunit rotation influences ATP and DNA binding. (A) Two interacting core ATPase domains are shown as ribbons (in cyan or green) along with the ball-and-stick models of R₀, E₁, E₂ and AMP–PNP. The E₁–R₀–E₂ triad acts as a clip to fasten two AMP–PNP binding surfaces at the neighbouring core ATPase domains. The E₁–R₀–E₂ triad gradually vanishes in the PfRad51 ring (B), in the SsoRadA left-handed filament (C) and in the overwound SsoRadA right-handed filament (D). Oxygen (red) and nitrogen (blue) atoms are shown. Selected hydrogen bonds are shown in pink lines in (A) and (B). (E) Effect of subunit rotation on DNA binding. R₀ is shown in deep pink. E₁ and E₂ are shown in cyan. The HhH (blue), L1 (pink) and L2 (green) motifs are indicated. The putative ssDNA binding paths, connecting all L1 and L2 motifs along the helical filaments, are indicated by black arrows. In the SsoRadA left-handed filament, all dsDNA binding HhH motifs are located at the outermost surfaces.

Figure 7.

EM images of the wild-type and mutant SsoRadA proteins. Negative staining EM images of SsoRadA wild-type, R83A, N85P, R83E and F73D proteins in the presence of AMP-PNP, Mg²⁺ and a ssDNA substrate (872 nucleotides in length). The results indicate that wild-type, R83A, N85P and R83E proteins are capable of forming filamentous structures. The wild-type (left-top panel) and N85P (left-middle panel) protein filaments often exhibited right-handed helices. In contrast, left-handed helices were often observed in the R83A protein filaments (right-top and right-middle panels). R83E proteins form filamentous structures with different lengths (left-bottom panel). The F73D point mutant proteins, defective in protein polymerization, form only protein aggregates with irregular morphology (right-bottom panel). Right- and left-handed helices are marked using white arrows.

Figure 8.

In vitro enzymatic activities of wild-type SsoRadA and SRM point mutants. (A) Time course analysis of D-loop formation was carried out as described in ‘Materials and Methods’ (16). (B) Quantitation of the D-loop time course experiments shown in (A). The amount of joined molecule or D-loop was quantified as the ratio of counts co-migrating with GW1 plasmid DNA to the total counts in each lane. A small faction (∼1/200) of total reaction mixture was used to determine the total counts. The efficiency of D-loop formation was calculated according to the molar ratio of joint molecules over total GW1 plasmid DNA. (C) The ATPase activities of SsoRadA proteins in the presence (+) or absence (−) of ssDNA substrate. The wild-type or mutant SsoRadA protein (2.4 µM) was first incubated with or without ΦX174 ssDNA (1 mM in nucleotides) in the presence of 1 mM Mg²⁺. ATP hydrolysis was initiated by addition of 1 mM [γ-³²P]ATP at 65°C. At different time points, 0.3 µl aliquots were withdrawn and spotted on thin layer chromatography paper as described previously (16). As expected, all five proteins examined here exhibited relatively low ATPase activities in the absence of ssDNA. (D) Kinetic analysis of the ssDNA-stimulated ATPase activities of wild-type or mutant SsoRadA proteins. The concentrations of wild-type, N85P, R83A, R83E and F73D proteins, denoted as [RadA], were 3.0, 2.0, 2.5, 5.0 and 5.0 μM, respectively. ATP hydrolysis was determined by addition of different amounts of [γ-³²P]-ATP (0.17, 0.34, 0.68 and 1.02 mM) at 65°C. Kinetic parameters (K_m and V_max) were determined by curve fitting using the Michaelis–Menten equation with a nonlinear least squares algorithm (Microcal Origin software). (E) EMSA analysis of wild-type or mutant SsoRadA proteins. A ssDNA substrate (50-mer; 1 μM) was incubated with increasing amount of protein (2.5, 5, and 10 μM). The resulting products were separated on a agarose gel and visualized by staining with ethidium bromide.

Figure 9.

Subunit rotation motif is essential for ScRad51 function in vivo. The yeast rad51 null mutant was transformed either with an empty control vector pYC2, with the pYC2-Rad51 expression vector for the wild-type Rad51, or with point mutant Rad51 proteins as indicated. Induction of these proteins was under the control of the RAD51 gene promoter. (A) MMS sensitivity assay was carried out as described in the ‘Material and Methods’. (B) Western blot analysis of the wild-type and point mutant Rad51 proteins. Total cell lysate was prepared, proteins were separated by SDS-PAGE and analyzed by Western blotting. Anti-Rad51 antibody (Santa Cruz Biotechnology, USA) and anti-tubulin (Invitrogen, USA) was used for detection of Rad51 and tubulin proteins. Tubulin was used here as a protein loading control. Final detection was performed using the ECL detection system. The emitted chemiluminescent light was recorded on X-ray films.

Figure 10.

Subunit rotation accounts for the structural transition of RecA right-handed filament from a compressed conformation (left panel) to a relaxed conformation (right panel). The N-terminal domain (NTD, in gray), the polymerization motif (PM, in yellow), ATPase domain (in green) and C-terminal domain (in gray) are shown as indicated, respectively. Ball-and-stick models of Ile27, Arg34 (R₀), Glu19 are shown along with oxygen (red) and nitrogen (blue) atoms. The side chain of Ile27 (in yellow) is responsible for filament assembly via interacting with a hydrophobic site in the ATPase domain of neighboring promoter. Arg34 forms a salt bridge with Glu19 in a compressed RecA helical filament (74 Å helical pitch, 33), and this salt bridge falls apart in a relaxed RecA helical filament (83 Å helical pitch, 29). The protein databank accession numbers of these two filaments are 1U94 and 2REB, respectively.

Figure 11.

The rotary motor hypothesis for RadA or Rad51 protein filaments.